ES cells are cells of cell lines established from an inner cell mass of blastocysts, and autonomously replicable in the presence of a leukemia inhibitory factor (LIF). ES cells can be differentiated into any type of cells (neuron, myocyte, vascular endothelial cell, erythrocyte, leukocyte, platelet, bone, cartilage, kidney, intestine, liver, pancreas, lung and the like) by changing culture conditions. Genomes of many kinds of animals have been decoded as the results of genome projects, and the information of their homologies with humans has been accumulated. The destruction of a particular gene in the stage of ES cells based on these information may elucidate a role (function) of the gene in cell differentiation, or growth of individual, homeostasis or the like. Specifically, a chimeric animal can be produced by injecting ES cells, in which a particular gene has been destructed, into normal host blastocysts to be mixed with the cells of the host embryos and returning the mixture to a uterus, and an animal, in which the particular gene has been destructed (knockout animal), can be produced by crossing the obtained chimeric animals. In addition, the effects of a compound on genes in various cells (organs) can be evaluated by applying the compound to ES cells. On the other hand, a use of normal cells obtained by differentiating ES cells enables cell therapy and regenerative medicine. As mentioned above, ES cells can be widely applied to studies in physiology, pharmacology, regenerative medicine and the like. However, in experimental animals, ES cells derived from only mouse (Evans M. J. et al., Nature 1981, 292: 154-156), rhesus monkey (Thomson J. A. et al., Proc. Natl. Acad. Sci. USA 1995, 92: 7844-7848), marmoset (Thomson J. A., et al., Cur. Top. Dev. Biol. 1998, 38: 133-165) and crab-eating monkey (Suemori H, et al., Methods Enzymol. 2003, 365: 419-429) have been established, and a rat ES cell has not been established yet.
Rat is a mammal having a size suitable for experiments (about 10 times the size of mouse), and it is advantageous in that (1) a drug can be easily administered to a minute blood vessel, (2) surgical and transplant experiments can be performed and (3) a large amount of tissue can be obtained.
Many human disease model rats have been developed and discovered. Rat is one of the most useful experimental animals, which is widely utilized in various fields including medicine. For the past 100 years, rat has been utilized for the studies in functions and the like as a model for cancer, cerebral nerve system, transplantation and human multifactorial disorder, and a vast amount of functional research resources has been accumulated. Particularly, the analysis of brain atlas is ongoing and abundant information of behavioral study in psychophysiology is available. As regards gene analysis, which is the weakest point as compared to mouse, too, since the analysis of rat genome is advancing, comparison with human at the gene level has been enabled, and rat has been drawing attention as an excellent experimental animal leading the post-genome. In fact, the Bioresource Area Subcommitte, the Life Science Subcommittee, the Subdivision on R&D planning and Evaluation, the Council for Science and Technology held in March, 2002 admitted that rat was one of the bioresources that should be enriched in Japan in the future, and that establishment of ES cells for the purpose of production of knockout rats was necessary.
Despite such utilities of rat and enrichment of genetic information therearound and the like, it has been extremely difficult to establish rat ES cells essential for the production of a genetically modified rat. For example, Iannaccone, P. M. et al. (Developmental Biology 1994, 163: 288-292) and the corresponding patent application WO 95/06716 describe that ES cells have been established from the rat strain PVG and chimeric rats have been produced. Thereafter, however, Brenin, D. et al. (Developmental Biology 1997, 185: 124-125) describe that the chimeric rats produced in Developmental Biology 1994, 163: 288-292 were produced by the contamination of mouse ES cells. Namely, it has been shown that they did not succeed in the establishment of rat ES cells and the production of chimeric rat. There are some other cases where the establishment of rat ES cell was attempted (see WO 99/27076 and JP 2002-176973 A), but all of them have not succeeded in the establishment.
As mentioned above, plural research groups have unsuccessfully tried to establish rat ES cell lines. In the INTERNATIONAL CONGRESS ON STEM CELLS held in Keystone in April 2004 in the United States, it was recognized that there had been no report on the establishment of rat ES cells all over the world. A key to the successful establishment of rat ES cells is the setting of culture conditions for the production of ES cells. While the conventional attempts have been made based on the establishment of and culture conditions for mouse ES cells, rat ES cells have not been established as yet. From these backgrounds, it is considered that ingenuity in the culture conditions is essential for the production of rat ES cells.